Antibiotic resistance and virulence profiles of Proteus mirabilis isolated from broiler chickens at abattoir in South Africa

Abstract Background Proteus mirabilis has been identified as an important zoonotic pathogen, causing several illnesses such as diarrhoea, keratitis and urinary tract infections. Objective This study assessed the prevalence of P. mirabilis in broiler chickens, its antibiotic resistance (AR) patterns, ESBL‐producing P. mirabilis and the presence of virulence genes. Methods A total of 26 isolates were confirmed as P. mirabilis from 480 pooled broiler chicken faecal samples by polymerase chain reaction (PCR). The disk diffusion method was used to evaluate the antibacterial susceptibility test, while nine virulence genes and 26 AR genes were also screened by PCR. Results All 26 P. mirabilis isolates harboured the ireA (siderophore receptors), ptA, and zapA (proteases), ucaA, pmfA, atfA, and mrpA (fimbriae), hlyA and hpmA (haemolysins) virulence genes. The P. mirabilis isolates were resistant to ciprofloxacin (62%) and levofloxacin (54%), while 8 (30.7%) of the isolates were classified as multidrug resistant (MDR). PCR analysis identified the blaCTX‐M gene (62%), blaTEM (58%) and blaCTX‐M‐2 (38%). Further screening for AMR genes identified mcr‐1, cat1, cat2, qnrA, qnrD and mecA, 12%, 19%, 12%, 54%, 27% and 8%, respectively for P. mirabilis isolates. The prevalence of the integron integrase intI1 and intI2 genes was 43% and 4%, respectively. Conclusions The rise of ciprofloxacin and levofloxacin resistance, as well as MDR strains, is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Furthermore, because ESBL‐producing P. mirabilis has the potential to spread to humans, the presence of blaCTX ‐M‐producing P. mirabilis in broilers should be kept under control. This is the first study undertaken to isolate P. mirabilis from chicken faecal samples and investigate its antibiotic resistance status as well as virulence profiles in South Africa.

The pathogenicity of P. mirabilis is associated with the ability to manifest virulence factors, such as the siderophore receptor ireA gene, haemolysing coding genes hpmA and hlyA, fimbriae coding genes atfA, pmfA, mrpA, ucaA, as well as the protease coding genes zapA and ptA (Li et al., 2016;Sanches et al., 2020).Previous studies reported 76% (38/50) of multidrug-resistant (MDR) Proteus species isolated from broilers in China (Li et al., 2022), while in Egypt a total of 22.8% (8/35) MDR Proteus species isolated from healthy and diseased ducks (Algammal et al., 2021).Polymerase chain reaction (PCR) method provides a quick, accurate, and reliable epidemiological screening tool for virulence and antibiotic resistance genes in bacterial pathogens (Algammal et al., 2021).
Beta-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures and are frequently prescribed antimicrobial agents all over the world in the treatment of infections caused by Gram-positive and Gram-negative bacteria (Bradford, 2001;Yarima et al., 2020).Pathogenic Gram-negative bacteria frequently produce Extended-Spectrum Beta-Lactamases (ESBLs), a genetic enzymatic component that contributes to their resistance to beta-lactam antimicrobial medicines (Pitout et al., 2005;Rao et al., 2014).ESBLs are plasmid-encoded enzymes that cause resistance to extendedspectrum cephalosporins, monobactams and aztreonams (CDC, 2019).
The P. mirabilis isolates have been described as having multiple acquired resistance genes encoding narrow-spectrum beta-lactamases and quinolones (Naas et al., 2003;De Champs et al., 2000;Girlich et al., 2020;Sanches et al., 2020).Hence, various genes encoding antibiotic resistance, including beta-lactamases and quinolones, should be screened, particularly for underreported bacteria such as P. mirabilis.
This study investigated the occurrence of P. mirabilis from faecal samples of broiler chickens collected in Mafikeng City, North-West Province, South Africa.Furthermore, this study documented the prevalence of P. mirabilis virulence genes as well as its phenotypic and genotypic antibiotic resistance patterns.

Sample collection
Following evisceration, 2400 chicken faeces samples were randomly collected from the caeca from 2016 to 2018 in four different abattoirs around Mafikeng City in the Ngaka Modiri Molema district of North-West Province, which is located between 25 • and 28 • south of the equator and 22 • to 28 • longitude east of the Greenwich Meridian (Ramatla et al., 2022).The faecal samples were pooled into a total of 480 samples, representing five separate broiler farms (Mileng et al., 2021).

Bacterial isolation and identification
For culture isolation of Proteus mirabilis from faeces 5 g of faecal sample was mixed with 10 mL of buffered peptone water (BPW Oxoid, Biolab, South Africa), homogenised for 2 min by vortexing and incubated for 18 to 24 h at 37

DNA extraction, PCR and sequencing
The bacterial genomic DNA was extracted using the Zymo Fun-

Antimicrobial susceptibility test
The phenotypic antibiotic resistance test was performed using the Kirby-Bauer (Disc diffusion) method to assess the antimicrobial sus-
Isolates that showed resistance to one or more of these antibiotics were confirmed as ESBL production.

Screening integron integrase genes by multiplex PCR assay
The presence of Int (intI1 and intI2) gene-encoding class 1 and 2 integrons was tested in all P. mirabilis isolates.The PCR reaction included a total reaction of 25 μL containing 12.5 μL of a 2X DreamTaq Green Master Mix (0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP, 4 mM MgCl 2 and loading buffer) (ThermoFisher Scientific, South Africa), 8.5 μL of nuclease-free water, 2.0 μL of the template DNA and 1.0 μL of each oligonucleotide primer.PCR primers and conditions for antibiotic resistance genes and class 1 and 2 integrons are shown in Supplementary Table S1.PCR reactions were performed using the ProFlex PCR System (Applied Biosystems, USA).

Data availability
The sequenced 16S rRNA gene of the 26 P. mirabilis isolates were deposited in GenBank under accession numbers ON832665-ON832690.

Identification of P. mirabilis
The P. mirabilis strains were isolated using classical microbiological tests on XLD media, which resulted in 46 suspected P. mirabilis colonies.A total of 26 isolates were positive on urease media with the production of pinkish-red colouration of the medium and P.

Antimicrobial susceptibility phenotypic profile
A total of 26 P. mirabilis isolates were further subjected to an antimicrobial susceptibility test to evaluate their resistance patterns (Table 1 mirabilis isolates (ON832672 and ON832681) were susceptible to all 14 antibiotics tested in this study.The distribution of antibiotic resistance on each P. mirabilis isolate is shown on the heatmap (Figure 1).

Multidrug-resistant P. mirabilis
A total of 8 (30.7%)P. mirabilis isolates were resistant to three or more antibiotic classes, which is an indication of a multidrug resistance pattern (Lv et al., 2022).Four (ABT 1, ABT 6, ABT 18 and ABT 26) isolates were resistant to four classes of antibiotics, followed by four (ABT 3, ABT 15, ABT 20, and ABT 21) isolates displaying resistance to three classes of antibiotics.

Distribution of ESBL genotypes
The ESBL genotype distribution is presented in

Detection of integron integrase genes
Twenty-six P. mirabilis strains were examined for the presence of two types of integrase genes.We observed the presence of the integrase gene (intI1) in 11 (42%) isolates, while 1 (C1HU 5) (4%) isolate harboured both intI1 and intI2 genes.

DISCUSSION
There are several published studies reporting P. mirabilis infections from human samples in South Africa as well as its antibiotic resistance patterns (Fourie et al., 2021;Irusen et al., 2021;Nana et al., 2021;Pitout et al., 1998).However, there is a scarcity of studies reporting the prevalence of P. mirabilis in animals.Hence, the current study sought to fill in such an information gap by investigating the occurrence of P. mirabilis in broiler chicken samples.South Africa.The microbiological culture-based approach and 16S rRNA gene sequencing were used to successfully identify 26 (5.4%) of the P. mirabilis strains out of 480 pooled faecal samples in this study.Pitout et al. (1998) identified P.
mirabilis as the cause of human patient wounds from hospitals in Cape Town, Durban, Soweto and Pretoria, while Botes (1964) identified these bacteria as the causative agent of an outbreak in calves.However, a number of investigations have been carried out in various nations to ascertain the incidence and presence of P. mirabilis in chicken samples.
An overall isolation rate of 7.07% for P. mirabilis was recorded in Shandong Province, China (Li et al., 2022).While P. mirabilis was isolated from chicken droppings in commercial poultry farms in Bangladesh with a prevalence of 39% (Nahar et al., 2014), while 46% of chicken carcasses were confirmed to have P. mirabilis infection in Pakistan (Ishaq et al., 2022).Based on empirical evidence, Proteus species are linked to human diseases, animal infections and the bacterial contamination of chicken products (Nahar et al., 2014).
Animal-derived P. mirabilis is a significant zoonotic pathogen that carries numerous virulence genes (Armbruster et al., 2019;Li et al., 2022).In the current study, P. mirabilis isolates contained the ptA, zapA, ucaA, pmfA, atfA, mrpA and hlyA genes.The ptA, atfA and mrpA genes were the predominant virulence genes detected in this study, with the highest detection rate of 100%.The findings of this study also demonstrated that the ireA, hlyA and hpmA genes were present in all P. mirabilis isolates, which is similar to the study conducted by Sanches et al. (2019), whereby all samples harboured the ireA and hpmA genes.
However, our results are in contrast with observations from previous studies, where very few isolates of P. mirabilis harboured these genes (Cestari et al., 2013;Lazm et al., 2018;Li et al., 2022;Uphoff & Welch, 1990).Variations may result from a difference in study time, geographic properties, sample types, sample sizes or identification methods.
Previously curable bacterial infections are now frequently untreatable or necessitate the use of antibiotics as a last resort (Sherchan et al., 2015;Wu et al., 2021).The P. mirabilis drug resistance has become increasingly serious in recent years (Li et al., 2022).This study revealed a higher antibiotic resistance rate against ciprofloxacin (62%) and the lowest rates of 8% for each of ertapenem and imipenem in the P. mirabilis isolates.The P. mirabilis was 39% resistant to gentamicin and 31% to amikacin from the perspective of aminoglycoside resistance.Ciprofloxacin (62%) was the antibiotic in this study with the highest level of fluoroquinolone resistance.This AR prevalence against ciprofloxacin is higher when compared to the findings of other  (Hasan et al., 2011;Nahar et al., 2014) and thus poses a public health concern.
The P. mirabilis isolates from broiler chickens are of public health concern since infections with these bacteria can result in failure to respond to the commonly used fluoroquinolone (ciprofloxacin), which increases mortality and delays treatment outcomes (Jamil et al., 2017).
Quinolone resistance is a current global issue in both human and veterinary medicine (Seo & Lee, 2019).Quinolone resistance genes mediated by the plasmids promote the spread of the multidrug resistance phenotype (Racewicz et al., 2022).The results obtained from this study revealed that one isolate was phenotypically and genotypically resistant to quinolones.The presence of the quinolone qnrA gene remains extremely rare in P. mirabilis (Girlich et al., 2020).However, in this study, quinolone-resistant genes, qnrA (54%) and qnrD (27%), were detected in P. mirabilis isolates.These results are very similar to those of Mokracka et al. (2012)   Brazil.One P. mirabilis ABT 12 strain harboured three (CIP, NA and LVX) antibiotic resistance and quinolone resistance genes (qnrA and qnrD).
The β-lactamase identified in Enterobacteriaceae has also been described in Proteus spp.(Girlich et al., 2020).The bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-8 , bla CTX-M-9 and bla CTX-M-25 enzymes are classified into five major phylogenetic groups (Girlich et al., 2020).We report the first detection of ESBL-P.mirabilis in broiler chickens in South Africa, char-acterised by both phenotypic and genotypic resistance.This study revealed that, of the 26 isolates of P. mirabilis tested using genotyping, 22 (84%) were identified as ESBL positive, while 8 (30.8%) were classified phenotypically as ESBL-producing.The genotypic method employing specific PCR amplification of resistance genes appears to be 100% sensitive and specific.There is a significant difference between phenotypic and genotypic methods, which emphasises the importance of molecular methods in interpreting antimicrobial resistance profiles.
Detection of resistance differs by phenotypic method due to its lower sensitivity and environmental factors on resistance incidence (Somily et al., 2015;Tewari et al., 2019).
The bla CTX gene was the highest detected gene (62% of ESBLproducing P. mirabilis isolates), followed by the bla TEM gene (58%) and the bla CTX-M-2 gene (38%).This is lower than the results reported in other countries, where 52% (147/282) and 12.86 % (75/583) ESBL genes were detected in P. mirabilis from chicken, pork and beef in Italy (Pagani et al., 2002) and in chicken, pork, beef and UTI-CA in Brazil, respectively (Sanches et al., 2023).Among our isolates, bla CTX-M , bla TEM and bla CTX-M-2 were the most commonly detected ESBL genotypes.
This agrees with other studies conducted in Korea, Italy and Brazil that reported the presence of genes from P. mirabilis (Ahn et al., 2017;Luzzaro et al., 2001;Sanches et al., 2023) but differs with the study conducted in Egypt (Shaaban et al., 2022), where the bla SHV , bla ampC and bla VIM-1 were the most detected genes.Multiple ESBL resistance genes may impart resistance to β-lactamases regardless of reduced expression of one or more of those genes (Gundran et al., 2019).
Most of the poultry isolates have more than one bla CTX-M group.
Since bla CTX-M has many homologous regions, this co-existence may result in the emergence of recombinant enzymes (Gundran et al., 2019).In total, five P. mirabilis (19.2%) have three types of bla CTX-M .
The bla CTX-M-9 gene was detected in three isolates in this study.Animals have been linked to bla CTX-M-9 -like enzymes either directly or indirectly in a number of countries, including Brazil (Sanches et al., 2023) and Korea (Song et al., 2011).The use of antimicrobials on farms is the major cause of the appearance of ESBL (CDC, 2019;Monyama et al., 2023;Wang et al., 2023).Our findings highlight a higher proportion of bla CTX-M -positive P. mirabilis isolates, further illuminating the propagation of this resistance gene among environmental P. mirabilis isolates.
It has been discovered that P. mirabilis is prone to spreading plasmidmediated ESBLs, including bla TEM -type derivatives that are active against expanded-spectrum cephalosporins (Biendo et al., 2005;Bonnet et al., 1999).In the present study, 58% bla TEM -positive P. mirabilis were detected by PCR.Proteus mirabilis has been described as harbouring a large number of bla TEM variants, including bla TEM-1 and bla TEM-2 penicillinases (Pagani et al., 2003).
The ESBLs and ampC beta-lactamases are produced by members of the Enterobacteriaceae in food-producing animals (von Tippelskirch et al., 2018).In this study, four isolates harboured the ampC gene.These results are different from the study conducted by Lin et al. (2019) in Northern Taiwan, whereby all isolates tested negative for the ampC gene.In the study conducted in Egypt, the prevalence of the ampC gene in P. mirabilis strains was 28.3% from the samples obtained from the hospital (Fam et al., 2013).In another study conducted in Egypt, one P.
Since MDR bacterial infections are associated with high death rates, emerging antibiotic resistance is currently recognised as one of the most critical public health issues.The P. mirabilis isolates harboured a high number of antibiotic-resistant strains in our study, with the highest proportion of drug resistance to 4 out of 4 classes of antibiotics.
A total of 8 (30.7%) isolates were resistant to ≥3 antibiotic classes, and four isolates were resistant to 4 classes of antibiotics.The MDR prevalence detected in the current study is lower compared to previously reported prevalence of 46% and 78.13% for P. mirabilis isolates in China (Li et al., 2022) and Brazil (Sanches et al., 2019), respectively.
However, the results in this study were higher than the reported prevalence of 28.7% antibiotic-resistant isolates in Northeast China (Sun et al., 2020).The MDR-associated intI1 gene was found in 35% of the isolates in the current study.One P. mirabilis isolate harboured the intI2 gene with four (CIP, GM, NA and LVX) antibiotic-resistant and quinolone-resistant genes (qnrA and qnrD).The prevalence of the integron integrase intI1 and intI2 genes was 42% and 4%, respectively.A study conducted by Mirzaei et al. (2021) showed a high prevalence of 60% for intI1 and 25% for intI2 genes compared to the results obtained in the current study.The high prevalence of MDR P. mirabilis isolates detected in this study raises real concern about substantial health risks since these strains may have a chance of contaminating food products and then spreading to humans.Future studies should seek to investigate the relationship between P. mirabilis isolates from animals and humans in order to understand the origin of antibiotic resistance development, as most antibiotics are used to treat infections in humans, while in animals such as chickens, they are used as growth promoters.

CONCLUSION
Human infections of P. mirabilis have already been reported by several studies in South Africa, demonstrating its public health and 'One Health' importance due to the development of antibiotic resistance (Fourie et al., 2021;Irusen et al., 2021;Nana et al., 2021;Pitout et al., 1998).In this study, P. mirabilis was isolated from broiler chicken faecal samples.We further confirmed that these P. mirabilis isolates possess virulence genes suggestive of a potential threat to food safety in chicken products.The findings of this study also paint a clear pic-ture of widespread antibiotic resistance to ciprofloxacin, amoxicillinclavulanic acid and gentamicin, which potentially poses a serious threat to public health.This study also detected three ESBL genes, bla CTX-M-8 , bla CTX-M , bla CTX-M-2 , bla TEM , bla OXA 2 and bla CTX-M-9 , in P. mirabilis isolates.This is the first study to detect P. mirabilis in chicken faeces in South Africa.Data generated in this study fills in the information gap about the zoonotic and 'One Health' significance of this bacterium in South Africa.This highlights the importance of formulation of a consolidated 'One Health' strategy to manage P. mirabilis infections by both the human and animal health sectors.

F
Heatmap showing the clustering of the antibiotic resistance profiles in the P. mirabilis isolates.Light blue and dark blue indicate absence and presence of antibiotic resistance respectively.
studies, whereby Kwiecińska-Piróg et al. (2013) reported only 40% AR in Poland.Hernandez et al. (2000) indicated that 16.2% of iso-lates were resistant to ciprofloxacin, while Ko et al. (2008) recorded 13.6% of P. mirabilis AR isolates in South Korea.The availability and accessibility of antimicrobial drugs such as streptomycin, ciprofloxacin, erythromycin, tetracycline and gentamicin in open markets to treat chickens (layer/broiler) represents a barrier to lowering antimicrobial resistance in poultry farms

F
Heatmap map showing clustering of virulence genes detected in 26 P. mirabilis isolates.Orange and green indicates the presence and absence of virulence genes, respectively.
To set up PCR assays for virulent genes, a total of 25 μL reaction mixture consisted of 12.5 μL of the PCR Master Mix (AmpliTaq Gold® DNA Polymerase, 0.05 units/L, Data analysis was carried out using Microsoft Excel 2016 (Microsoft Corporation, Redmond, DC, USA).The sequenced 16S rRNA gene of the 26 isolates was aligned to nucleotide sequences available in GenBank and identified by comparing them with those available in the National Centre for Biotechnology Information database (NCBI) using BLASTn (http://www.ncbi.nlm.nih.gov/BLAST/).The heatmap plots of the virulence and antibiotic resistance profile were generated using ChipPlot (https://www.chiplot.online/#). ).
Virulence genes, antibiotic resistance phenotypes, antibiotic resistance genes and integrons of P. mirabilis.
Santos et al. (2014)27% of isolates consisting of qnrD in Europe.Chloramphenicol resistance genes [chloramphenicol acetyltransferase (cat1 19% and catII 12%)] were also detected in this study.Similar findings were reported bySantos et al. (2014), where they detected the cat1 gene in 14.3% of P. mirabilis isolates in TA B L E 2